1. Field of the Invention
The present invention relates generally to a device for temperature controlled chemical and/or bio-chemical reactions. More particularly, the present invention relates to real-time detection of Polymerase Chain Reaction (PCR).
Many applications such as microbiology, genetic disease diagnostics, forensic, food science only have small amount of DNA for analysis, which is very difficult to detect. Polymerase chain reaction became a very valuable technique which is capable of producing large amount of copied DNA fragments from minute amounts of DNA samples, for both sequencing and genotyping applications.
During PCR process, the solution undergoes temperature cycles to create copy of the original DNA fragment in each cycle. Each temperature cycle consists of generally three steps: (1) Denaturation (˜95° C.); (2) Annealing (˜50° C.); (3) Extension/Elongation (˜70° C.).
Two important factors are critical to the PCR tests: the ability for the samples in the microplate holding the samples to reach their set point temperature quickly so the whole test can be completed in reasonable time frame, and the ability of the reaction module to maintain temperature uniformity among array of microwells for each set point temperature.
2. Description of the Related Art
For purposes of screening, statistical analysis, or large scale assay project, it is highly desirable to process many samples at the same time under similar test conditions. Most common PCR sample tray (microplate) is constructed with a solid top frame holding many microwells arranged in 2-D pattern, such as 12×8 (a total of 96 wells) format, 24×16 (a total of 384 wells) format. The microwell usually has conical profile (FIG. 8A-B) for ease of insertion and removal from the thermal block, in which temperature is controlled, as described in U.S. Pat. No. 6,015,534 (Atwood, The Perkin-Elmer Corporation). The thermal block has matching machined conically shaped cavities to accommodate the sample wells. The thermal block is usually attached to a thermoelectric module for controlled heating and cooling, or it has channels machined near the bottom to allow heating or cooling fluid to pass through to realize temperature control.
The thermal block of such design is fairly complex and expensive to make, the microplate also has to have its conical shaped wells matched perfectly to the cavity geometry to get the uniform heating/cooling desired. The other drawbacks include: heat transfer to the sample could take a long time since it has to travel substantial distance upwards from the bottom of the thermal block to reach top portion of the microwell; this also introduces non-uniform heating/cooling in the sample solution from top to bottom since the bottom part will reach the set point temperature much earlier than the top portion. Such design is also far from optimal from optical performance standpoint, since both excitation and emission light have to travel through the depth of the microwell which results in significant signal attenuation.
There have been incremental improvements over such design. One example, as described in U.S. Patent Application Pub. No. 2010/0055743 A1 (Banerji, Bio-Rad Laboratories, Inc.), the thermal block was trimmed to reduce thermal mass to improve response time. Such incremental improvement came with extra costs for more complex thermal block design and manufacturing. It didn't resolve the non-uniform heating/cooling issue at different depths of the microwell.
Another design available in the market is glass capillary tube design. Slender glass tubes loaded with sample solutions are placed onto the thermal control module in circular pattern, convective heating/cooling is utilized by blowing temperature controlled air stream to this glass tube ring array. The glass tube has to be very thin to obtain good heat transfer which makes it fairly fragile; it also has to have small cross-section for the same reason, which makes it difficult to inject the sample solution. In addition, this method has the challenge of scaling up to accommodate large number of samples.
There have been other ideas to further enhance the PCR thermal module performance. One example was described in U.S. Pat. No. 5,459,300 (Kasman), in which a thermally conductive compliant layer was added between the microplate and the heating surface, with the desire to accommodate various existing microplate bottom geometries (flat, U-shaped, V-shaped). Such compliant layer, even with the addition of thermally conductive fillers, usually has very poor thermal properties compared to metals such as aluminum and copper, and it also introduces additional thermal interface, all these result in slow response time. Furthermore, heat transfer is very sensitive to variation of the thickness of the compliant layer when it is under vertical load, a parameter very difficult to control under such embodiment, resulting in non-uniform heating and response time among microwells. In addition, most microplate designs do not assume heat transfer through microwell bottom, which could further deteriorate the solution's thermal performance.
The other approach, as described in U.S. Pat. No. 7,074,367 (Lurz, et al.), used a static PCR microplate coupled with a sample block, while allowing thermostated blocks (set at different temperatures/profiles) to make contact to its bottom surface. How to effectively make the contact interface thermally optimal (low contact resistance, uniform across the whole surface) is a significant challenge. This solution still suffers the large thermal mass encountered in conventional design, resulting in slow thermal response and long cycle time.
There are other flat bottom microplate designs, one example as described in U.S. Pat. No. 6,232,114 (Coassin, et al., Aurora Bioscience Corporation) to address mainly optical accessibility challenges, other than the thermal response and uniformity problems that the PCR process encountered.
Accordingly, there is a need in the art to establish a device that would address cost, thermal response, and uniformity for the PCR process.